Combined ultrasound and tissue plasminogen activator (rt-PA) therapy, or sonothrombolysis, has been shown to improve recanalization in patients with acute ischemic stroke. Effective methods of enhancing thrombolysis have been examined in an attempt to reduce the dosage of the thrombolytic agent and reduce the risk of hemorrhagic events. We have investigated the synergistic effect of rt-PA and 120-KHz ultrasound on thrombolysis using in vitro porcine and human whole blood clot models. In our ongoing studies, we have demonstrated that significant enhancement of thrombolysis correlates with the presence of stable cavitation and this type of gentle bubble activity can be sustained using an intermittent infusion of a contrast agent. In addition, we have shown that inertial cavitation, which elicits broadband acoustic emissions, is counter-productive for enhanced thrombolysis. Rather, the most effective form of bubble activity is stable cavitation, which elicits ultrasonic subharmonic generation. Importantly, we have shown encapsulation of rt-PA in a contrast agent specifically targeted to clot. These preliminary data strongly support the central hypothesis of our proposal that ultrasound enhances thrombolysis primarily via mechanical mechanisms. To test this hypothesis we propose to investigate three Specific Aims: In Aim #1, we will develop a dual-element annular array transducer to facilitate simultaneous 120-kHz pulsed ultrasound exposure and passive cavitation detection in vitro and in vivo. In Aim #2, we will demonstrate the efficacy of 120-kHz ultrasound-enhanced thrombolysis through in vivo studies in a porcine intracerebral hemorrhagic stroke model using fluorescently labeled rt-PA-loaded liposomes or gas contrast agents and neuropathologic examination. As a novel approach in Aim #3, we will investigate the potential of echogenic liposomes to deliver rt-PA and nitric oxide, a bioactive gas, near an intravascular clot in an ex vivo porcine carotid model. Vascular reactivity and the degree of rt-PA leakage across the vascular endothelium will be assessed to clarify the potential risks for sonothrombolysis in the presence of gas contrast agents. Successful completion of these studies will contribute significantly to our long-term goal to develop a sonothrombolysis system that delivers and enhances thrombolytic therapy in the cerebral vasculature and rapidly restores perfusion after ischemic stroke. PUBLIC HEALTH RELEVANCE: Our long-term objective is to develop a transcranial, ultrasound-enhanced thrombolysis system that minimizes the risk of intracranial hemorrhage, increases the number of stroke survivors, improves long-term prognosis, and reduces health care costs. The development of the agents and techniques listed in this proposal would have far reaching implications in improving directed therapeutic treatment of stroke.